Epithelial cells use cell-autonomous defense mechanisms to eliminate intracellular pathogens. These defense mechanisms are often activated or augmented by the cytokine Interferon-gamma (IFN?). We recently discovered that IFN?-primed mouse cells employ ubiquitin systems to mark Chlamydia-containing vacuoles (?inclusions?) for destruction. We reported that a subset of IFN?-inducible Immunity Related GTPases (IRGs) ? the so-called GKS proteins ? bind to inclusions, recruit ubiquitin E3 ligases and thereby promote the decoration of inclusions with ubiquitin. Ubiquitin-marked inclusions are subsequently destroyed and intracellular Chlamydia eliminated by the murine host cell. Our preliminary data show that IFN?-primed human cells also deposit ubiquitin on inclusions. Therefore, both humans and mice evolved IFN?-inducible ubiquitination pathways that target inclusions. However, our data indicate that mouse and human IFN?-inducible ubiquitination pathways are mechanistically distinct: while GKS proteins are essential for inclusion ubiquitination in mouse cells, GKS protein-encoding genes are absent from the human genome. We have further shown that the mouse-adapted pathogen Chlamydia muridarum can block the function of GKS proteins and is therefore resistant to ubiquitination in mouse cells but is susceptible to inclusion ubiquitination in human cells. The situation is reversed for the human-adapted pathogen Chlamydia trachomatis: we found that C. trachomatis inclusions label ubiquitin-positive in IFN?-primed mouse cells but remain largely devoid of ubiquitin in IFN?-primed human cells. These results show that C. trachomatis is uniquely adapted to its human host and has evolved mechanisms to avoid a human-specific ubiquitin-mediated response. The goal of this proposal is twofold: in Aim1 we will characterize this newly discovered IFN?-inducible human defense pathway and identify human factors critical for inclusion ubiquitination. To be able to monitor a successful human host response, we will infect human cells with the mouse pathogen C. muridarum. We will employ customized siRNA and CRISPR/Cas libraries to identify human genes responsible for the attachment of ubiquitin to inclusions. In Aim2 we will seek to identify C. trachomatis anti-immune factors that interfere with IFN?-induced induced ubiquitination of inclusions in human cells. We will use an arrayed and validated C. trachomatis mutant library to screen for C. trachomatis mutants that are susceptible to inclusion ubiquitination in human cells. Knowledge gained from this proposal will open up avenues for the development of novel therapeutics that aim to boost the inherent ability of the human host to detect and eliminate C. trachomatis and potentially other intracellular pathogens.